Since accumulation of misfolded proinsulin in the ER causes pancreatic β cells failure and results in permanent neonatal diabetes, understanding how misfolded proinsulin is recognized and retro-translocated from the Endoplasmic Reticulum (ER) lumen to the cytosol for degradation is an important biomedical issue. We have developed a unique experimental approach to characterize the kinetics of proinsulin retro-translocation (RT). In this experimental approach, wild type proinsulin and a mutant proinsulin that ends up misfolded in the ER are purified, labeled with BODIPY dye and individually encapsulated in reconstituted ER microsomes (RRM) containing all or a selection of lumenal proteins and small molecules. Retro-translocation is then assessed by monitoring the movement of the BODIPY-labeled proinsulin from the lumen of the RRM microsomes into the cytosol where it would be degraded in the cell. BODIPY-proinsulin entry into the cytosol from the RRM is detected by the quenching of BODIPY fluorescence that occurs when BODIPY-specific antibodies in the cytosolic medium bind to the dye. Because quenching is immediate upon BODIPY-proinsulin exposure to the antibodies, this approach directly measures the real-time rate of proinsulin retro-translocation.

Since our reconstituted ER microsomes contain only individual or a specific combination of lumenal chaperones, this approach will allow us to systematically assess the functional involvement of specific lumenal chaperones in recognizing and retro-translocating misfolded proinsulin for degradation.

Chaperone-assisted co-translational protein folding in the ER We have developed a novel system to spectroscopically monitor secretory protein (prolactin) co-translational folding in the ER and characterize the functional involvement of lumenal chaperones in the folding process. We will characterize the extent of FRET-detected folding as a function of both nascent chain length and also lumenal content (using reconstituted ER microsomes) to determine which lumenal proteins and small molecules affect nascent chain folding, in what order, and whether they act singly or in concert with others.